Flavin-containing monooxygenases (FMO) oxygenate numerous drugs and other xenobiotics. Caucasians and Asians do not express functional pulmonary FMO2 but up to 50% of African individuals and 3-10% of Hispanics express at least one allele coding for catalytically active enzyme (FMO2.1). Mycobacterium tuberculosis, the causative agent in tuberculosis (TB), possesses a similar flavin monooxygenase (EtaA) that catalyzes S-oxygenation of second-line anti-tuberculosis drugs to the metabolite toxic to the bacteria. Ethionamide (ETA) is one of a class of drugs increasingly used due to development of resistance to first-line therapeutic regimens. FMO2.1 is more efficient in the firstS-oxygenation of ETA to the sulfenic acid than EtaA, but bacterial enzyme is more efficient in the second S-oxygenation to the sulfinic acid having bactericidal activity. The sulfenic acid is capable of redox-cycling with glutathione producing oxidative/nitrative stress and toxicity. There is a fundamental knowledge gap in understanding the role of human FMO2 in ETA action. The highest incidence of individuals with active FMO2.1 and the highest incidence of TB co-occur in Sub-Saharan Africa where resistance to front-line TB drugs is of great importance. Our central hypothesis is that individuals having active FMO2.1 will have reduced ETA efficacy in inhibition and killing of M. tuberculosis and enhanced oxidative/nitrative stress and pulmonary toxicity, issues not previously considered. Understanding the effect of genotype on ETA metabolism and thus on M. tuberculosis will enable improved drug selection and utilization. The central hypothesis will be tested by three integrated specific aims employing innovative tools under the guidance of a powerful investigative team: 1) Test, in vitro, the efficacy of FMO and EtaA in metabolism of ETA, bacterial killing and lung cell damage employing bacterial cell culture and transfected human lung cells; 2) Employ a newly developed Fmo1/2/4 triple knockout mouse to assess in vivo the contribution of FMO and EtaA in ETA metabolism, therapeutic efficacy and lung toxicity; and 3) Construct and utilize a transgenic humanized FMO2.1 mouse to better model human response. The approach is innovative: it creates a highly unique environment capitalizing on FMO and TB expertise and facilities, uses the first Fmo knockout mouse available and extends and enhances the model by creation of the first humanized FMO mouse; for the first time absolute quantification by mass spectrometry is used to quantify individual FMOs. The proposed research is significant with the potential for high impact: it will lead to better matching of individuals with appropriate drug therapy. Relevance to Public Health: The WHO has termed TB a global health emergency. Two billion people (1/3 the world's population) are infected with M. tuberculosis; in the next 12 years another billion will be infected and 30 million will die. The outcome of the studies and work facilitated by this grant is expected to provide guidance for physicians seeking to identify and administer the drug most likely to maximize individual drug response and minimize TB drug toxicity.